1. Field of the Invention
The present invention relates to the spectroscopy of colloidal particles that are used to advantage in monitoring chemical binding assays. The invention pertains to colloids that are much smaller than a wavelength of light and show the phenomena of resonance at certain wavelengths which: (1) enhances optical absorbance above the background of liquid samples; and (2) introduces a strong particle shape dependence into the optical absorbance spectrum.
2. Description of Related Art
Chemical binding assays have been used in the laboratory for a wide range of analyses including the detection of genetic material, ligand-receptor interactions for therapeutic drug screening, and immunoassays for tumor markers, hormones, and infectious disease detection. The basis of a chemical binding assay is the existence of highly specific chemical binding between two molecular components. Such binding events can involve two or more complex macromolecules such as a protein ligand and its receptor, an adhesion molecule and its target binding molecule, or two polynucleotides, or can involve interaction between a complex molecule such as an antibody or lectin with a smaller molecule known as a hapten. The important point is a "lock in key" fit between the two components that results in a relatively high affinity of one component for the other.
The typical strategy of a chemical binding assay is to use one component of a binding pair to assay for the presence and concentration of the second component of the pair. Most commonly one of the components of the pair is immobilized on a solid substrate so that it can be readily separated from the other material used in the analysis. That is, a sample solution containing an unknown quantity of the second component of the binding pair is added to the immobilized first component so that the two can bind together. Then the immobilized component (and the bound second component) is removed from the sample solution, washed to remove impurities, and then assayed for the presence of the unknown second component. This process is known as a bound-free separation
It should be appreciated that while chemical binding is often explained in terms of a "binding pair," the actual situation is frequently more complex. Although any particular chemical binding occurs between two components, when assaying an unknown macromolecule by means of a chemical binding assay, more than two different components may easily be involved. For example, assume that the unknown material is a protein "antigen" produced by a disease organism. A good way of identifying the protein antigen is by binding a specific antibody to the antigen. The antibody is a special protein with a specific binding site that is complementary to a specific site or epitope on the protein antigen. The point is that most proteins are large enough to contain more than one epitope. If the unknown material is a bacterium or a virus, there may be hundreds or thousands of epitopes. This is to say, although an antibody is highly specific to the antigen or virus, many different antibodies may be specific to different sites on the antigen or virus. Therefore, the strategy of the chemical binding assay may advantageously involve more than one pair of chemical binding components. For example, two or more antibodies specific to different epitopes on a macromolecule or cellular component can be used in a single chemical binding assay. In the case of an assay designed to detect a specific nucleic acid sequence, multiple binding components, each specific to a different subsequence, can advantageously be used.
In many laboratory analyses that depend on chemical binding assays, the test is automated so that little human participation is required. This necessarily requires more or less complex fluid handling equipment which, in addition to dispensing accurate and precise fluid volumes, must also perform the separation of the immobilized component from the sample solution, the bound-free separation. Bound-free separation is generally cumbersome, and requires specialized equipment such as centrifuges or microplate washers.
In order to eliminate the bound-free separation step and reduce the time and equipment needed for a chemical binding assay, so-called "homogeneous assay" methods have been developed. Many of these methods still immobilize one component of the binding pair, but manage to detect the presence of the second component of the binding pair without a bound-free separation. Examples of homogeneous, optical methods are the EMIT method of Syva, Inc. (Sunnyvale, Calif.), which operates through detection of fluorescence quenching, the laser nephelometry latex particle agglutination method of Behringwerke (Marburg, Germany), which operates by detecting changes in light scatter, the LPIA latex particle agglutination method of Mitsubishi Chemical Industries, the TDX fluorescence depolarization method of Abbott Laboratories (Abbott Park, Ill.), and the fluorescence energy transfer method of Cis Bio International (Paris, France).
Of the optically based, homogeneous methods, those that involve particle aggregation have been preferred for general use because there is no limitation as to analyte size or molecular weight. In the TDX method of fluorescence depolarization, the analyte molecular weight must be low (e.g. hapten analyses) the assay operates by detecting differences in molecular rotation between a small free analyte and that same analyte bound to a comparatively large binding member. In the EMIT system there is a steric chemical limitation on the ability of analytes to cause detectable fluorescence quenching--excessively large analytes are unable to effect quenching. Particle binding assays do not have these inherent molecular weight limitations, and have been used for analyses with molecular weights ranging from a few hundred Daltons to a few million Daltons.
Opposing the molecular weight advantage, optically monitored, particle binding assays have a well-known restriction which relates to nonspecific chemical binding. That is, the aggregation of large particles (e.g. of the order of one micrometer in diameter) is easily detected by changes in optical extinction, but these particles present large surface areas that are prone to nonspecific chemical binding. Nonspecifically bound material can cause false readings and generally decreases the accuracy of the analysis. The relation between particle size and nonspecific binding can be appreciated from the following examples. A one-micrometer particle, such as is used in many visually read particle aggregation chemical binding assays, presents a surface area that has room for a nonspecifically bound monolayer of several thousand macromolecules, approximately. The LPIA optical extinction method utilizes polystyrene (latex) particles with diameters slightly less than the wavelength of visible light, i.e., 250 nanometers. These particles have the capacity to nonspecifically bind only several hundred macromolecules. If particles are further reduced in size to be far smaller than a wavelength of visible light, say approximately 20 nanometers to 50 nanometers, the particles will have room for only one to ten nonspecifically bound macromolecules. With larger particles, opportunity for nonspecific binding to particles, and nonspecific cross-linking between particles, is clearly great. Unfortunately, when the particles are much smaller than a wavelength of light, the optical signals produced by these particles are usually greatly reduced.
It is known that metal colloids may at time produce strongly colored solutions; see "Full-color photosensitive glass," Stookey, S. D., Beal, G. H. and J. E. Pierson, J Appl. Phys. 49: 5114-23 (1978). Leuvering et al. (U.S. Pat. No. 4,313,734) discloses that metallic colloidal particles can be used in protein binding assays with detection representing a color change. This reference advocates the use of a wide range of metals and metal oxides. Further, this reference advocates the use of a mixed colloid with particles of a variety of sizes and many doublet and other aggregate particles.
In one example using silver, Leuvering et al. demonstrates the use of a gray-yellow-green colloidal suspension with multiple, complex, bound A gray-yellow-green colloidal silver suspension is indicative of a mixture of small (approximately 10 nm diameter), single particles (yellow color) and large, preformed, particle aggregates up to diameters of approximately 130 nm (gray-green color) (see Absorption and Scattering of Light by Small Particles, Bohren, C. F. and Huffman, D. R., p.372, John Wiley and Sons, New York, 1983).
Laserna et al. ("Effect of Substrate Optical Absorption on Surface-Enhanced Raman Spectrometry on Colloidal Silver," Laserna, J. J., Cabalin, L. M., and Montes, R., Anal. Chem. 64:2006-09 (1992)), has described methods of making aggregate free, colloidal silver suspensions, and has stressed that a single absorption peak at approximately 400 nm (which yields a pure yellow color) is indicative of a highly monomeric suspension, and that broad absorption bands at longer wavelengths between 450 nm and 900 nm are indicative of aggregates even when these bands are not the region of maximum absorption.
Thus, the potential advantage of less nonspecific binding with very small particles is at least partly lost by the presence of much larger particles and aggregates in the assay of Leuvering et al. Further, this assay method appears to be largely a heterogeneous assay that depends on bound-free separations. Until the present invention it has not been possible to greatly reduce nonspecific binding through the use of extremely small particles because the small particles produced optical signals that were greatly diminished as compared to larger particles. These optical signals were generally inadequate for use in homogeneous assays where the sample solution is not removed by washing so that other substances present in the sample solution produce confounding background signals.